1. Technical Field
The invention relates to a method for isolating and purifying nucleic acids and/or oligonucleotides from a biological sample, to the use of the isolated or purified nucleic acid and/or oligonucleotide for transfecting cells and also for the production of an agent for the treatment of genetic disorders, to a composition suitable for the isolation or purification method and also to the use of potassium acetate and a silica gel-like support material for isolating endotoxin-free nucleic acids and/or oligonucleotides or nucleic acids and/or oligonucleotides with reduced endotoxin content.
2. Description of the Background Art
The quality of isolated nucleic acids is becoming increasingly important. Highly pure nucleic acid fractions, i.e. fractions from which, if possible, all other cell components such as, for example, endotoxins, have been removed, play a central part in gene therapy or in transfecting cells of eukaryotic or also prokaryotic origin. Consequently, in the past few years methods or measures which allow the isolation of nucleic acids from biological sample material with high purity have increasingly been published. The established methods essentially make use of the use of affinity and/or anile exchange chromatography materials and also of ionic detergents or also diluted solutions of higher alcohols. For example, according to WO95/21177 the fractions of interest are subjected to an affinity chromatography or a chromatography on an inorganic solid phase, the latter preferably in the presence of a non-ionic detergent, in order to remove endotoxins and are then further purified by means of anion exchange chromatography. A two-stage chromatography method of this kind, however, is time- and material-consuming and therefore is more academically valuable. According to another method (WO95/21178) a complicated anion exchange chromatography is likewise absolutely necessary in order to remove residues of a complex salt solution added beforehand.
Furthermore, it has been known for some time that DNA plasmids from complex biological samples of eukaryotic or prokaryotic origin can be isolated by binding to silica gel in the presence of chaotropic salts such as, for example, guanidine hydrochloride (M. A. Marko et al., Analyt. Biochem. 121, (1982) 382-287; EP 0 389 063). However, these methods are not suitable for obtaining low-endotoxin or endotoxin-free nucleic acid fractions. Thus it has been possible to show, for example, that the measures according to Marko et al. (1982) lead to an endotoxin content of more than 10,000 Upper mg of DNA. Such an endotoxin-rich DNA fraction is unsuitable for transfecting cells in applications of gene therapy.